Several scientific and patent publications are referenced in this patent application to describe the state of the art to which the invention pertains. Each of these publications is incorporated by reference herein, in its entirety.
The family Picornaviridae includes at least five genera: cardiovirus, aphthovirus, enterovirus, rhinovirus, hepatovirus and a possibly a newly proposed genus paraenterovirus. The genus enterovirus (EV) is subdivided into five groups: the polioviruses (PV), coxsackieviruses groups A (CVA) and B (CVB), echoviruses (ECV) and the numbered EV.
The picornaviral genome is a single stranded, positive sense RNA molecule approximately 7,500 to 8,300 nucleotides (nts) in length. The genome is organized into a 5′ nontranslated region (5′NTR), a polyprotein-coding region, a 3′NTR, and a terminal poly A tract. In all picornaviridae a small protein, VPg, is covalently bound to the 5′ terminal-pUpUp of the genome. The viral coding region can be subdivided into the P1, P2 and P3 regions. In the non-human picornaviridae (cardio- and aphthovirus) the P1 region is preceded by an “L” region that encodes for a L proteinase. The P1 region encodes the four capsid proteins (VP14), while the P2 and P3 regions encode seven nonstructural proteins essential for the picornavirus life-cycle.
Picornaviridae 5′ Nontranslated Region. The 5′NTR of the sequenced enteroviruses (EV) is remarkably constant in size (711-747 nts). Sequence analysis has demonstrated the existence of multiple regions of highly conserved nucleotide identity. Multiple stem-loop (SL) structures (or domains) exist within this region of the genome. Additionally, there is evidence for the existence of tertiary structural elements (pseudoknots) within the EV 5′NTR. Similarly, for the hepatoviruses and non-human picornaviridae (cardio- and aphthovirus) conserved secondary structures have also been predicted to exist within the 5′NTR.
Studies of PV 1-3, CVB 1 and, most recently, CVB 3 have demonstrated that the 5′NTR contains genomic elements necessary for replication, translation, and determinants of virulence. The initial 98 nucleotides (nts) of the 5′NTR from PV have been demonstrated to play a role in viral RNA replication. A ribonucleoprotein (RNP) complex at the 5′ end of the PV NTR has been shown to be important for RNA replication. This RNP complex comprises the initial 98 nts of the 5′NTR (which fold into cloverleaf-like structure), the viral protein 3CD, and poly C binding protein 2 (PCBP2). For the other human picornaviridae a similar folding motif is predicted for the initial 84-98 nts.
The existence of a cis-acting genomic element within the 5′NTR of PV, in conjunction with trans-acting cellular proteins, is required for efficient translation of the protein coding region. This element, termed the internal ribosome entry site (IRES), is a discontinuous region spanning approximately from nts 140 to 620 of the PV 5′NTR. Evidence for the requirement of SLs II, IV, V and VI in PV translation has been provided. Stem-loop III has been proven to be nonessential for cap-independent translation. For representatives from each of the genera of the picornavirus family, a region of approximately 450 nts within the 5′NTR is required for cap-independent internal initiation of protein synthesis. Studies focusing on the downstream portion of the IRES have identified specific sequences and/or higher order structures that directly influence the ability of the PV IRES to efficiently initiate translation.
Picornaviridae tissue and species tropism. A major determinant of tissue and species tropism for the picornaviridae is the presence or absence of a viral receptor on the cell surface. For the picornaviridae this has been best studied for the PV. Poliovirus has a distinct species and tissue tropism, infecting only primates. In primates PV has a restricted tissue tropism; replicating only in pharynx, gut and neurons within certain regions of the central nervous system. However, PV has been shown to bind to tissues that do not support PV replication. Additionally, poliovirus receptor (PVR) RNA and protein has been shown to be expressed in tissues that are not sites of PV replication. These and other findings indicate that PV tissue tropism is not governed solely by the presence of the PVR in tissues.
Evidence is beginning to accumulate that viral 5′NTR-host protein interactions may be the level at which restriction of picornavirus species and tissue tropism occurs. The strongest support for the role of 5′NTR-cellular interactions as determinants of host range restriction comes from work by Shiroki et al. (J. Virol. 71: 1-8, 1997). PV1 (Mahoney strain) mutants within SLII at nts 128-134 were found to replicate well in primate cells but not in murine cells of TgPVR mice. SLII mutants demonstrated high neurovirulence in monkeys and low neurovirulence in mice. The IRES dependent translation of the SLII mutants was found to be blocked in Tg mouse kidney cells (TgSVA) and mouse neuroblastoma cells (NS20Y) but not in HeLa cells. A follow-up study showed that SLII mutation revertants that recovered IRES function in a TgSVA cell-free translation system also recovered neurovirulence in mice (Ishii et al., J. Virol. 72: 2398-2405, 1998). These studies strongly suggest that an additional determinant of species or host range tropism is the interaction between the IRES and host factor(s). This restriction may be the result of host-restricted expression PV IRES function. These studies were performed using artificially altered 5′NTRs and therefore may not reflect the actual events that occur naturally. Evidence for host/tissue range restriction in a naturally-occurring picornavirus secondary to the IRES heretofore has not been available.
Virulence of CVB and other Picornaviruses. The group B coxsackieviruses (CVB) are responsible for a myriad of clinical syndromes involving almost every organ system that range from febrile exanthems to myocarditis and meningoencephalitis. It is widely accepted that the CVB are a major, if not the predominant, cause of viral myocarditis in humans. However, it is not yet know what elements of the CVB determine their ability to cause diseases in humans.
The enteroviral capsid has been shown to contain determinants contributing to the pathogenic phenotype of CVB4, CVB3, and the polioviruses (PVs). However, the sites determining the virulence phenotypes of these viruses do not co-localize to a single capsid region or even a single capsid protein. Determinants have been found in all four capsid proteins and are not necessarily located at surface-exposed residues of the virion.
For instance, a noncardiovirulent antibody escape mutant derived from the highly cardiovirulent CVB3/H3 strain was found to contain a single amino acid substitution (Asn3Asp) at position 165 of VP2 (Knowlton et al., J. Virol. 70: 7811-7818, 1996). When Asp165 was substituted for the Asn165 in VP2 of the parental cardiovirulent CVB3 strain, the myocarditic phenotype was significantly attenuated. Conversely, a change to Asn165 in VP2 of the antibody escape mutant reverted this strain to the cardiovirulent phenotype.
Specific nucleotide(s) within the 5′NTR are also known to alter the virulence phenotype of the PVs (reviewed by Minor, J. Gen. Virol. 73: 3065-3077, 1992), CVB1 (Rinehart et al., J. Virol. 71: 3986-3991, 1997), and CVB3 (Tu et al., J. Virol. 69: 4607-4618, 1995). A U→C mutation at nt 234 within the CVB3 5′NTR results in attenuation of the cardiovirulent phenotype in mice. Replacement of the cardiovirulent CVB3/M or CVB3/20 5′NTRs with that from CVB3/0 attenuates the resultant viruses for myocarditis (Tu et al., 1995, supra; Lee et al., J. Med. Virol. 52: 341-347, 1997). Subsequent analysis of multiple clinical CVB3 isolates as well as other enteroviruses demonstrated that nt 234 is always U regardless of the cardiovirulence phenotype of the virus, consistent with 234C being an artificial mutation.
Zhang et al. (J. Med. Virol. 41: 129-137, 1993) isolated an attenuated CVB3 strain (p14V1) following multiple passages of cardiovirulent CVB3/Nancy in human dermatofibroblasts. Sequence analysis of the 5′NTR revealed a single nucleotide change at position 690 (A→U). Insertion of 690U into the cardiovirulent parental virus did not alter the myocarditic phenotype, demonstrating that this mutation does not affect the cardiovirulence phenotype. Following passage of p14V1 in scid mice hearts, a revertant to cardiovirulence was isolated (Cameron-Wilson et al., Clin. Diagn. Virol. 9: 99-105, 1998). Sequence comparison of the 5′NTR and capsid coding region of this revertant to the attenuated p14V1 and cardiovirulent CVB3/Nancy strains suggested that amino acid 155 in VP1 might play a role in attenuation; however, this has not yet been demonstrated.
The studies of genomic determinants of virulence for the foregoing and the majority of enteroviruses have relied on strains engineered by physiochemical or biologic means in the laboratory. As a result, it remains undetermined whether the anomalies in the 5′NTRs of those strains are clinically relevant determinants of virulence, i.e., in naturally occurring EVs.